Molecular Imaging of the Immune-Suppressive TME in Prostate Cancer

Treatment with immune checkpoints inhibitors has had unprecedented responses in turning some deadly cancers into chronic diseases. Despite these successes, 80% of cancers including prostate cancer (PCa) have complex tumor microenvironments (TMEs) that are resistant to immune checkpoint inhibitors. In the US, PCa is the second leading cause of death from cancer in men. The vast majority of men dying of PCa succumb to metastatic castration-resistant disease. PCa frequently exhibits hypoxia. Understanding the role of the TME including hypoxia, metabolism, fibroblasts and the extracellular matrix (ECM), in creating an immune suppressive and metastasis permissive TME can lead to treatments to induce an immune reactive TME responsive to CIT, and reduce mortality from PCa. Imaging methods that provide spatio-temporal information on mechanisms that create barriers to immune cell infiltration to improve the outcome of CIT in PCa are urgently needed to accelerate progress in this field. Here we will apply molecular and functional imaging to understand the role of hypoxia, metabolism, prostate fibroblasts (PFs) and prostate cancer associated fibroblasts (PCAFs) in creating an immune suppressive TME, and in PCa invasion and metastasis. Studies will be performed using human and mouse PCa cells, PFs and PCAFs, preclinical human PCa models, syngeneic PCa models in immune competent mice, and human PCa TMAs. A HIF-1a inhibitor, PX-478, used in clinical trials (NCT00522652) will be used to determine changes in the tumor immune microenvironment and metabolism. In Aim 1, our MR-compatible cell perfusion system that allows careful control of oxygenation will be used to understand the role of PFs and PCAFs in increasing the ability of PCa cells (DU-145, PC-3, RM1 and TRAMP-C2) to invade and degrade ECM under normoxia and hypoxia, and alter metabolism. In Aims 2 and 3 we will use castrate-resistant human PCa xenografts (DU-145 and PC-3) in severe combined immune deficient (SCIO) mice, and syngeneic tumors (RM-1 and TRAMP-C2) in immune competent mice. In Aim 2, in vivo bioluminescent imaging (BLI) of PCa models derived from cells with luciferase under control of the hypoxia response element (HRE) will be combined with PD-L1 PET imaging, ex vivo BLI and second harmonic generation (SHG) microscopy, immunostaining, mass spectrometry imaging and molecular characterization to understand the relationship between tumor hypoxia and and PD-L1 expression, CAF numbers, metabolism, collagen 1 (Col1) fiber patterns and immune cells in these regions. Validation of observations made in tumor models will be performed in human PCa TMAs immunostained for HIF-1a to detect hypoxia and PD-L1/PD-1 and T-cells, a-SMA (smooth muscle actin) to detect CAFs, and SHG microscopy for Col1 fiber patterns. In Aim 3, we will determine the effects of PX-478 on PD-L1 expression, Col1 fibers, metabolism, immune cells, growth, and metastasis. These studies will expand the identification of achievable targets such as hypoxia, CAFs, and metabolism in improving the outcome of CIT in PCa. Justification of animal models In keeping with the NIH emphasis on in vitro systems, we will use our intact cell perfusion system to understand the effects of hypoxia on PFs, PCAFs, and PCa cells on ECM invasion, degradation and metabolism in Aim1. However, the complexity of the tumor microenvironment including the ECM necessitates further validation of these studies with solid tumors in vivo that will be derived from PCa cells engineered to express luciferase under hypoxia for detection with bioluminescent imaging. The use of tumor models will allow noninvasive imaging of hypoxia and PD-L1 to understand the relationship between hypoxia and PD-L1 that will be combined with ex vivo analysis of tumor models and human PCa TMAs in Aim 2. Tumor models will also be required in Aim 3 where we will inject the HIF-1a inhibitor, PX-478, in mice to determine the effects of inhibiting hypoxia in altering the TME and Project Number: 1R01CA287368-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Zaver Bhujwalla | Institution: JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD | Award Amount: $477,674 | Activity Code: R01 | Study Section: Imaging Probes and Contrast Agents Study Section[IPCA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11376021